The present invention relates to the field of plant molecular biology, more particularly to regulation of gene expression in plants.
Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. Where expression in specific tissues or organs is desired, tissue-preferred promoters may be used. Where gene expression in response to a stimulus is desired, inducible promoters are the regulatory element of choice. In contrast, where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. Additional regulatory sequences upstream and/or downstream from the core promoter sequence may be included in the expression constructs of transformation vectors to bring about varying levels of expression of heterologous nucleotide sequences in a transgenic plant.
Frequently it is desirable to express a DNA sequence in particular tissues or organs of a plant. For example, increased resistance of a plant to infection by soil- and air-borne pathogens might be accomplished by genetic manipulation of the plant""s genome to comprise a tissue-preferred promoter operably linked to a heterologous pathogen-resistance gene such that pathogen-resistance proteins are produced in the desired plant tissue.
Alternatively, it might be desirable to inhibit expression of a native DNA sequence within a plant""s tissues to achieve a desired phenotype. In this case, such inhibition might be accomplished with transformation of the plant to comprise a tissue-preferred promoter operably linked to an antisense nucleotide sequence, such that expression of the antisense sequence produces an RNA transcript that interferes with translation of the mRNA of the native DNA sequence.
Thus far, the regulation of gene expression in plant roots has not been adequately studied despite the root""s importance to plant development. To some degree this is attributable to a lack of readily available, root-specific biochemical functions whose genes may be cloned, studied, and manipulated. Genetically altering plants through the use of genetic engineering techniques and thus producing a plant with useful traits requires the availability of a variety of promoters. An accumulation of promoters would enable the investigator to design recombinant DNA molecules that are capable of being expressed at desired levels and cellular locales. Therefore, a collection of tissue-preferred promoters would allow for a new trait to be expressed in the desired tissue. Several genes have been described by Takahashi et al. (1991), Plant J. 1:327-332; Takahashi et al. (1990), Proc. Natl. Acad. Sci. USA 87:8013-8016; Hertig et al. (1991), Plant Mol Biol. 16:171-174; Xu et al. (1995), Plant Mol Biol. 27:237-248; Capone et al. (1994), Plant Mol Biol. 25:681-691; Masuda et al. (1999), Plant Cell Physiol. 40(11):1177-81; Luschnig et al. (1998), Genes Dev. 12(14):2175-87; Goddemeier et al. (1998), Plant Mol Biol. 36(5):799-802; and Yamamoto et al. (1991), Plant Cell. 3(4):371-82 to express preferentially in plant root tissues.
Metallothioneins (MT""s) are proteins or polypeptides that bind and sequester ionic forms of certain metals in plant and animal tissues. Examples of such metals include copper, zinc, cadmium, mercury, gold, silver, cobalt, nickel and bismuth. The specific metals sequestered by MT""s vary for the structurally distinct proteins/polypeptides occurring in different organisms. Plants appear to contain a diversity of metal-binding MT""s with the potential to perform distinct roles in the metabolism of different metal ions. In plants, MT""s are suggested to have roles in metal accumulation, metal intoxication, and embryogenesis.
Typically, MT""s and MT-like proteins are Cys-rich proteins, characterized by the presence of Cys-Xaa-Cys motifs suggesting the capability of binding metal ions. Further categories of MT-like proteins have been proposed on the basis of the predicted locations of Cys residues and designated types 1 and 2. In type 1 there are exclusively Cys-Xaa-Cys motifs, whereas in type 2 there is a Cys-Cys and a Cys-Xaa-Xaa-Cys pair within the N-terminal domain. The type 1 motif has been implicated in the binding and sequestration of copper.
Several metallothionein-like plant genes have been isolated, including those from pea, maize, barley, mimulus, soybean, castorbean and arabidopsis. See Robinson et al. (1993) Biochem J. 295: 1-10. Sequences expressed in roots have been reported to be isolated from pea, as described in Evans et al. (1990) FEBS Lett 262:29-32. A maize root MT gene has been described in U.S. Pat. No. 5,466,785; though this sequence is also expressed leaves, pith and seed, as described in de Framond (1991) FEBS Lett 290:103-106.
Thus, isolation and characterization of tissue-preferred, particularly root-preferred, promoters that can serve as regulatory regions for expression of heterologous nucleotide sequences of interest in a tissue-preferred manner are needed for genetic manipulation of plants. Furthermore, isolation and characterization of sequences involved in metal-binding and accumulation are needed for influencing metabolism of metals in plants.
Compositions and methods for regulating expression of heterologous nucleotide sequences in a plant are provided. Compositions comprise novel nucleotide sequences for promoters that initiate transcription in a root-preferred manner. More particularly, a transcriptional initiation region isolated from a plant metallothionein gene, is provided. A method for expressing a heterologous nucleotide sequence in a plant using the transcriptional initiation sequences disclosed herein is provided. The method comprises transforming a plant cell with a transformation vector that comprises a heterologous nucleotide sequence operably linked to one of the plant promoters of the present invention and regenerating a stably transformed plant from the transformed plant cell. In this manner, the promoter sequences are useful for controlling the expression of operably linked coding sequences in a root-preferred manner.
Downstream from and under the transcriptional initiation regulation of the promoter will be a sequence of interest that will provide for modification of the phenotype of the plant. Such modification includes modulating the production of an endogenous product, as to amount, relative distribution, or the like, or production of an exogenous expression product to provide for a novel; function or product in the plant.
Also provided are compositions and methods for expressing metallothionein genes in plants, plant cells, and plant tissues. The compositions comprise nucleotide sequences for the expressed region of the MT gene, which comprise the nucleotide sequences encoding the metallothionein polypeptide. These sequences are useful in transforming plants for tissue-preferred or constitutive expression of metallothionein. Such sequences find use in modulating levels of metal ions in plants and plant tissues.
Expression cassettes comprising the sequences of the invention are provided. Additionally provided are transformed plant cells, plant tissues, and plants.